Methods for preparing and delivering adjuvant compositions

ABSTRACT

Methods are provided for preparing and delivering an adjuvant for vaccines including lecithin and a polymer. The polymer is preferably polyacrylic acid-based and the delivery method involves administering a vaccine, including an antigen and the adjuvant, to a mucosal surface.

CROSS REFERENCE TO RELATED APPLICATION

[0001] This application is a divisional of application Ser. No.09/884,201, filed Jun. 19, 2001.

FIELD OF THE INVENTION

[0002] The present invention pertains to methods for preparing anddelivering vaccine onto mucosal surfaces by, for example, oral orintranasal administration or with an antigen, topically or parenterallyas an injectable.

BACKGROUND OF THE INVENTION

[0003] Mucosal delivery of vaccines has been underutilized because ofthe problems associated with effectively delivering the vaccine antigensto the mucosal surface and to the underlying mucosal lymphoid tissue.Since mucosal surfaces are the port of entry of the majority of theinfectious agents (Sabin, A. B., Vaccination at the portal of entry ofinfectious agents. Dev Biol Stand 33:3-9, 1976) it is important to thehealth of an animal to have developed a strong protective antibody andcell-mediated immune response at the portal of entry. This is best donewith an adjuvant and delivery system that targets vaccine antigens toeither the mucous membranes of the oral cavity, gut, nose, rectum, orvagina. Because this is not commonly done with an injectable vaccine, itwould be advantageous to have a vaccine adjuvant delivery compositionthat would adsorb the vaccine onto the mucosal surface, and then,following absorption, be brought in contact with mucosal-associatedlymphoid tissue.

[0004] For example, oral administration of a vaccine against a gutpathogen may engender a stronger immune response against such pathogensby eliciting the production of secretory immunoglobulin A antibodies atthe mucosal site. This happens when the vaccine is presented to thegut-associated lymphoid tissue (O'Hagen, D, Oral Delivery of Vaccines:Formulation and Clinical Pharmacokinetic Considerations 1992, Clin.Pharmacokinet. 22 (1): 1-10). Likewise, administration of vaccineagainst an upper respiratory pathogen may be most effective if deliveredto the mucosal-associated lymphoid tissue in the oral cavity or nasalpassages. Interestingly, administration of antigens induces a mucosalimmune response not only at the site of antigen application, for examplethe oral mucosa, but also at other mucosal sites such as the nasalmucosal (Mestecky, J I, The Common Mucosal Immune System and CurrentStrategies for Induction of Immune Responses in External Secretions. JClin Immunol. 7 (4): 265-76).

[0005] Vaccinating large numbers of animals, such as cattle, swine andpoultry, is extremely labor intensive and expensive. Each individualanimal has to be handled at the time of vaccination in order to injectthe animal with the vaccine. Most often the vaccine must be administeredto the animal at least twice, and sometimes three or more times. Itwould be advantageous in terms of time and expense if the vaccine couldbe administered, simultaneously, with feed or water to a large number ofanimals.

[0006] Another advantage of targeting the vaccine to mucosal surfaces isthat the vaccine can stimulate a protective immune response in thepresence of circulating antibody that interferes with parenterallyinjected vaccines (Periwal, S B, et.al., Orally administeredmicroencapsulated reovirus can bypass suckled, neutralizing maternalantibody that inhibits active immunization of neonates. J Virol 1997(Apr 71(4): 2844-50).

[0007] Adjuvant systems to enhance an animal's immune response to avaccine antigen are well known in the art. Likewise, systems for thedelivery of vaccine and drugs to mucosal surfaces are known in the art.Different methods have been described to protect the vaccine antigen anddrugs from degradation by stomach acid and digestive enzymes and toadsorb the antigen to the mucosal surface. Often these adjuvants anddelivery systems include mixing the antigen with one or more components.

[0008] Examples of prior art adjuvants include the following.

[0009] U.S. Pat. No. 4,917,892, Speaker et al, issued Apr. 17, 1990,describes a topical delivery system comprising a viscous carriercontaining a dissolved or dispersed active agent and active agentmicroencapsulated within a semi permeable anisotropic salt film which isthe emulsion reaction product of a) a partially lipophilic, partiallyhydrophilic, polyfunctional Lewis acid or salt thereof in aqueousmedium, such as carboxymethylcellulose, an alkali metal salt ofpolyacrylic acid or cross linked polyacrylic acid/polyoxyethylene, withb) a Lewis base or salt thereof in a water-immiscible, slightly polarorganic solvent for the base, such as benzalkonium chloride, andpiperidine. U.S. Pat. No. 5,132,117, Speaker et al., issued Jul. 21,1992, discloses a microcapsule with an aqueous core, capsular, ionicstabilized anisotropic Lewis salt membrane formed from the interfacialreaction product of an emulsion of an aqueous solution of awater-soluble, hydrophilic polymeric Lewis acid or salt thereof with anon-aqueous solution of a lipophilic Lewis base or salt thereof. TheLewis base may be stearylamine, piperidine, or benzalkonium chloride andthe Lewis acid may be carboxymethylcellulose, polyacrylic acid, orpolyacrylic acid/polyoxyethylene copolymer, for example.

[0010] U.S. Pat. No. 4,740,365, Yukimatsu et al., issued Apr. 26, 1988describes a sustained-release preparation applicable to mucous membranesin the oral cavity. The preparation consists of an active ingredient ina mixture of a polymer component (A) comprising one or more polymersselected from polyvinylpyrrolidone, polyvinyl alcohol, polyethyleneglycol, alginic acid or a salt thereof, and an alternating copolymer ofmaleic anhydride and methyl vinyl ether and a polymer component (B)comprising one or more polymers selected from polyacrylic acid and asalt thereof. Polymer component (A) and (B) are in a ratio of 95:5 to5:95 by weight. The preparation is layered with the active ingredientand may have optional conventional carriers and additives.

[0011] U.S. Pat. No. 5,451,411, Gombotz et al., issued Sep. 19, 1995,describes a delivery system for a cationic therapeutic agent whereuponalginate has been cross-linked in the presence of the therapeutic agentand polyacrylic acid to obtain a sustained release composition for oraldelivery.

[0012] U.S. Pat. No. 5,352,448, Bowersock et al., issued Oct. 4, 1994,describes an oral vaccine formulation comprising an enzymaticallydegradable antigen in a hydrogel matrix for stimulation of an immuneresponse in gut-associated lymphoid tissues. The hydrogel pellets arepreferably synthesized by polymerizing methacrylic acid, in the presenceof methylene bis-acrylamide and ammonium persulfate and sodiumbisulfite.

[0013] U.S. Pat. No. 5,674,495, Bowersock et al., issued Oct. 7, 1997,describes a vaccine composition for oral administration comprising analginate gel in the form of discrete particles. The alginate gel maycontain a polymer coating such a poly-l-lysine to enhance stability andto add a positive charge to the surface.

[0014] U.S. Pat. No. 4,944,942, Brown et al., issued Jul. 31, 1990,describes an intranasal vaccine for horses, which may comprisepolyacrylic acid cross linked polyallyl sucrose, sold as Carbopol 934P,combined with polyoxyethylene sorbitan mono-oleate and sorbitanmonolaurate, preferably at 7.5 to 15 volume percent based on the totalvolume of the formulation, as an adjuvant.

[0015] U.S. Pat. No. 5,500,161, Andrianov et al., issued Mar. 19, 1996,describes a method for the preparation of microparticles, and theproduct thereof, that includes dispersing a substantially waterinsoluble non-ionic or ionic polymer in a aqueous solution in which thesubstance to be delivered is also dissolved, dispersed or suspended, andthen coagulating the polymer together with the substance by impactforces to form a microparticle. Alternatively, the microparticle isformed by coagulation of an aqueous polymeric dispersion through the useof electrolytes, pH changes, organic solvents in low concentrations, ortemperature changes to form polymer matrices encapsulating biologicalmaterials.

[0016] U.S. Pat. No. 6,015,576, See et al., issued Jan. 18, 2000,describes a method that comprises orally administering lyophilizedmultilamellar liposomes containing the antigen wherein the liposomepreparation is contained in a pill form or within an enterically coatedcapsule. Such an enteric coating may be composed of acrylic polymers andcopolymers.

[0017] U.S. Pat. No. 5,811,128, Tice et al., issued Sep. 22, 1998,describes a method, and compositions for delivering a bioactive agent toan animal entailing the steps of encapsulating effective amounts of theagent in a biocompatible excipient to form microcapsules having a sizeless than approximately ten micrometers and administering effectiveamounts of the microcapsules to the animal. A pulsatile response isobtained, as well as mucosal and systemic immunity. The biocompatibleexcipient is selected from the group consisting ofpoly(DL-lactide-co-glycolide), poly(lactide), poly(glycolide),copolyoxalates, polycaprolactone, polyorthoesters and poly(beta-hydroxybutyric acid), polyanhydrides and mixtures thereof.

[0018] U.S. Pat. No. 5,565,209, Rijke, issued Oct. 15, 1996, describesoil-free vaccines comprising polyoxypropylene-polyoxyethylene polyolsand an acrylic acid polymer as adjuvant constituents for injectablevaccines.

[0019] U.S. Pat. No. 5,084,269, Kullenberg, issued Jan. 28, 1992,describes an adjuvant, comprised of lecithin in combination with acarrier which may be selected from the group consisting of non-edibleoil such as mineral oil and edible triglyceride oils such as soybeanoil, for an injectable vaccine.

[0020] U.S. Pat. No. 5,026,543, Rijke, issued Jun. 25, 1991, disclosesoil-free vaccines which contain polyoxypropylene-polyoxyethylene polyolsas well as an acrylic acid polymer as adjuvanting constituents.

[0021] U.S. Pat. No. 5,451,411, Gombotz et al, issued Sep. 19, 1995,discloses alginate beads as a site specific oral delivery system forcationic therapeutic agents designed to target the agents to the luminalside of the small intestine. Enhanced bioactivity of therapeutic agentsreleased from the alginate is attributed to the ability of polyacrylicacid to shield the agents from interaction with lower molecular weightfragments of acid treated alginate.

[0022] U.S. Pat. No. 5,567,433, Collins, issued Oct. 22, 1996, disclosesa method of producing liposomes useful for encapsulating and deliveringa wide variety of biologically active materials. The method involves theformation of a liposome dispersion in the absence of an organic solventor detergent, one or several cycles of freezing and thawing, anddehydration to form a lipid powder. The powder is hydrated in thepresence of a biologically active material to encapsulate it in theliposomes.

[0023] U.S. Pat. No. 5,091,188, Haynes, issued Feb. 25, 1992, discloseswater-insoluble drugs are rendered injectable by formulation as aqueoussuspensions of phospholipid-coated microcrystals.

[0024] The current invention relieves the problems found in the priorart by providing a simpler composition and easier method of formulationas well as providing easier delivery.

[0025] It is therefore a primary object of the present invention toprovide a novel vaccine delivery system and adjuvant.

[0026] It is another object of the present invention to provide avaccine adjuvant which is especially useful for mucosal delivery ofvaccines.

[0027] It is a further object of the present invention to provide avaccine adjuvant that allows vaccine to be administered in animals' feedor water.

[0028] It is yet a further object of the present invention to provide avaccine adjuvant that may be conveniently administered, practicallysimultaneously, to a large number of animals.

[0029] It is still a further object of the present invention to providea method of incorporating antigen into a vaccine delivery system withminimal damage to vaccine epitopes.

[0030] It is a further object of the present invention to provide asimple method and means of manufacturing a vaccine delivery system thatdoes not require harsh conditions such as elevated temperatures ororganic solvents or detergents.

[0031] These and other objects, features, and advantages will becomeapparent after review of the following description and claims of theinvention which follow.

SUMMARY OF THE INVENTION

[0032] The present invention concerns an adjuvant composition thatincludes lecithin and a polymer that is preferably an acrylic polymer orcopolymer. The preferred acrylic polymer is a polyacrylic acid polymer.

[0033] The lecithin and polymer form a matrix or net-like structurewhich is effective in trapping or encapsulating vaccine antigen.Further, the strong mucoadhesive and adsorptive properties of thepolymer and lecithin combination enhances the adsorption of vaccineantigen onto mucosal surfaces. Further, the lecithin compositionenhances absorption (Swenson, E S and W J Curatolo, © Means to EnhancePenetration (2) Intestinal permeability enhancement for proteins,peptides and other polar drugs: mechanisms and potential toxicity.Advanced Drug Delivery Reviews. 1992. 8: 39-92) that helps bring theantigen in contact with the underlying lymphoid tissue.

[0034] The adjuvant composition of this invention makes it possible tovaccinate via a mucosal surface, such as oral cavity, gut, nasal,rectal, or vaginal surfaces. The vaccine may be administered by pill ortablet form, a paste form or in fluid form using a dropper or needlelesssyringe. This adjuvant composition allows a method of vaccination viafood and/or water. The composition can be used traditionally as aninjectable as well.

[0035] In accordance with the invention, there are provided methods formaking and delivering the above-mentioned composition.

[0036] Further features and advantages of the present invention will beset forth in, or apparent from, the detailed description of preferredembodiments thereof which follows.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0037] The present invention concerns a vaccine adjuvant which, whenadmixed with an antigen or hapten and administered into a human oranimal, will induce a more intense immune response to the antigen thanwhen the antigen is administered alone. The present invention alsoconcerns vaccines comprising an antigen or group of antigens and a noveladjuvant herein described which comprises a combination of lecithin anda polymer, which is preferably an acrylic polymer. As will appear, thepresent invention specifically concerns methods of making and using theforegoing adjuvants and vaccines.

[0038] The present invention offers the advantage of allowingapplication of a vaccine directly to a mucosal surface. In doing so, thevaccine stimulates a protective immune response which helps preventinterference from circulating maternal antibodies that may be present ina newborn or infant, for example.

[0039] “Antigen” is herein defined as a compound which, when introducedinto an animal or a human, will result in the formation of antibodiesand cell-mediated immunity.

[0040] “Adjuvant” is herein defined as a compound or compounds that,when used in combination with specific vaccine antigens in formulations,augment or otherwise alter or modify the resultant immune responses.

[0041] “Vaccine” is herein defined as a composition of antigenicmoieties, usually consisting of modified-live (attenuated) orinactivated infectious agents, or some part of the infectious agents,that is administered, most often with an adjuvant, into the body toproduce active immunity.

[0042] The antigen for use in this invention may be any desired antigenfalling within the definition set forth above. Antigens are commerciallyavailable or one of skill in the art is capable of producing them. Theantigenic moiety making up the vaccine can be either a modified-live orkilled microorganism, or a natural product purified from a microorganismor other cell including, but not limited to, tumor cells, a syntheticproduct, a genetically engineered protein, peptide, polysaccharide orsimilar product, or an allergen. The antigenic moiety can also be asubunit of a protein, peptide, polysaccharide or similar product. Theantigen may also be the genetic antigens, i.e., the DNA or RNA thatengenders an immune response. Representative of the antigens that can beused according to the present invention include, but are not limited to,natural, recombinant or synthetic products derived from viruses,bacteria, fungi, parasites and other infectious agents in addition toautoimmune diseases, hormones, or tumor antigens which might be used inprophylactic or therapeutic vaccines and allergens. The viral orbacterial products can be components which the organism produced byenzymatic cleavage or can be components of the organism that wereproduced by recombinant DNA techniques that are well known to those ofordinary skill in the art. Because of the nature of the invention andits mode of delivery it is very conceivable that the invention wouldalso function as a delivery system for drugs, such as hormones,antibiotics and antivirals.

[0043] The lecithin for use in this invention is any lecithin or, forinstance, lecithin lipoidal material, such as phosphotidylcholine, thatcan be used to form liposomes. Phospholipids, lysophospholipids,glycolipids and neutral lipids comprise the typical composition oflecithin. Lecithins are molecules that, when completely hydrolyzed,yield two molecules of fatty acid, and one molecule each of glycerol,phosphoric acid, and a basic nitrogenous compound, which is usuallycholine. The fatty acids obtained from lecithins on hydrolysis areusually, but not limited to, oleic, palmitic, and stearic acids. Thephosphoric acid may be attached to the glycerol in either an α- or theβ-position, forming α-glycerophosphoric acid or β-glycerophosphoricacid, respectively, and producing the corresponding series of lecithinswhich are known as α- and β-lecithins.

[0044] Commercial lecithin is obtained by extraction processes from eggyolk, brain tissue, or soybeans. Ovolecithin (vitelin) from eggs andvegilecithin from soybeans, as well as purified lecithin from calf'sbrains have been used as emulsifiers, antioxidants, and stabilizers infoods and pharmaceutical preparations. Commercial lecithin may beobtained from a variety of sources, for example Central Soya (FortWayne, Ind.). One of ordinary skill in the art would be able todetermine an appropriate lecithin for a desired application.

[0045] The polymer is preferably an acrylic polymer, which is anypolymer or copolymer that contains an acrylic moiety. Examples ofsuitable acrylic polymers include, but are not limited to polyacrylicacid, methacrylic acid, methacrylate, acrylamide, acrylate,acrylnitrile, and alkyl-esters of poly acrylic acid. Examples of acryliccopolymers are poly(acrylamide-co butyl, methacrylate),acrylic-methacrylic acid, acrylic-acrylamide and poly(methacrylate).Examples of commercially available acrylic polymers include, Carbopol(B. F. Goodrich Co., Cleveland, Ohio), Carboset, (B. F. Goodrich Co.,Cleveland, Ohio), Neocryl (Avecia, Inc., Wilmington, Del.), and Eudragit(Rohm Tech, Inc., Malden, Mass.).

[0046] Certain acrylic polymers, such as Carbopol and Eudragit, maybenefit from the inclusion of a cross linker, such as a polyalkenylpolyether or an alkyl sucrose, which is effective in binding thepolymers. Both Carbopol and Eudragit are available in commercialformulations that include polyalkenyl polyether or alkyl sucrose crosslinkers. The most preferred acrylic polymer for use in this invention ispolyacrylic acid (Carbopol), with or without a polyalkenyl polyethercross linker. One of ordinary skill in the art would be able todetermine an appropriate acrylic polymer for a desired application.

[0047] Examples of non-acrylic polymers that are suitable are polyvinylacetate phthalate, cellulose acetate phthalate, methylcellulose,polyethylene glycol, polyvinyl alcohol, and polyoxyethylene

[0048] The method of manufacturing the adjuvant of this invention firstinvolves hydrating the lecithin and polymer by suspending from about0.0001-10% by weight/volume dry lecithin and from about 0.0001-10% byweight polymer in saline or water. The preferred concentrations oflecithin and polymer are 0.001-1.0% each by weight/volume. The twocomponents may be mixed together using conventional methods, such as,for example, a Waring Blender, emulsification equipment or amicrofluidizer. Surfactants (emulsifiers) may be added to aid in themixing or emulsification of the lecithin and polymer. Suitable syntheticdetergents are well known to those of ordinary skill in the art.Examples of appropriate surfactants include polyoxyethylene sorbitanmonooleate, sorbitan monolaurate, sodium stearate, non-ionicether-linked surfactants such as Laureth®4 and Laureth®23, alkyl sulfatesurfactants, alkyl alkoxylated sulfate surfactants,alkylbenzenesulphonates, alkanesulphonates, olefinsulphonates,sulphonated polycarboxylic acids, alkyl glycerol sulfonates, fatty acylglycerol sulfonates, fatty oleyl glycerol sulfates, alkyl phenolethylene oxide ether sulfates, paraffin sulfonates, alkyl phosphates,isothionates such as the acyl isothionates, N-acyl taurates, fatty acidamides of methyl tauride, alkyl succinamates and sulfosuccinates, mono-and diesters of sulfosuccinate, N-acyl sarcosinates, sulfates ofalkylpolysaccharides, branched primary alkyl sulfates, alkyl polyethoxycarboxylates, and fatty acids esterified with isethionic acid andneutralized with sodium hydroxide. Further examples are given in SurfaceActive Agents and Detergents (Vol. I and II by Schwartz, Perry andBerch), the disclosure of which is expressly incorporated herein byreference. Suitable nonionic detergent surfactants are generallydisclosed in U.S. Pat. No. 3,929,678, Laughlin et al., issued Dec. 30,1975, at column 13, line 14 through column 16, line 6, incorporatedherein by reference. If included, the emulsifier should be added in aconcentration ranging from about 0.001-0.05% by volume of the mixture.

[0049] Although the invention itself has adjuvant properties, it mayalso be used in combination with other adjuvants such as, but notlimited to, saponins, fractions of saponins, synthesized components ofsaponins, ISCOMS, muramyl dipeptide and analogues, pluronic polyols,trehalose dimycolate, amine containing compounds, cytokines andlipopolysaccharide derivatives. The addition of another adjuvant may aidin the stimulation of a mucosal immune response. If included, additionaladjuvants may be present in a concentration of up to about 10% by weightof the composition, with less than about 1% by weight being preferred.

[0050] The invention may also include one or more probiotics. Probioticsare bacteria or microorganisms that are beneficial to the health of theindividual or animal. Examples of commonly used probiotics include, butare not limited to, various beneficial strains of Lactobacillus,Bifidobacterium, Streptococcus, etc. If present, each of the organismsshould be administered in a concentration ranging from about 10³ to 10⁸CFU each.

[0051] In addition to all of the above, as is well understood by thoseskilled in the art, other minors can be employed to make the basiccomposition more pharmaceutically and/or cosmetically elegant. Forexample, dyes can be added at very minor levels as can diluents such asalcohol, buffers, stabilizers, wetting agents, dissolving agents,colors, etc. With the exception of diluents such as alcohols which areused at higher levels, the levels of these minors are generally not morethan 0.001% to 1.0% by weight.

[0052] If desired, the adjuvant components may be sterilized byautoclaving prior to the hydration step. It has also been found thatautoclaving and/or microwaving the components may improve theirsuspending ability. The vaccine antigen may be added after formation ofthe adjuvant, or at the time the time of hydration of the adjuvantcomponents. If in tablet form, the antigen may be mixed with drycomponents of the adjuvant invention along with other excipientsnecessary for tablet formation. Examples of appropriate types of vaccineantigens include killed or attenuated bacterial, viral, parasitic, orsubunits of these organisms, or genomic vaccine antigens, for example,DNA.

[0053] The relative concentration of the components, including theantigen, may be determined by testing the formulations in animalsstarting with a low dose of the formulation and then increasing thedosage while monitoring the immune response. The followingconsiderations should be made when determining an optimal dose, e.g.,breed, age, size and the presence or absence of interfering maternalantibodies.

[0054] A concentration of an attenuated viral vaccine will compriseabout 10³ to 10⁹ TCID₅₀ per animal. Preferable the amount will be fromabout 10⁴ to 10⁷ TCID₅₀ per animal. The concentration of killed antigenor subunit antigen may range from nanogram to milligram quantities ofantigen with about 1 microgram to 1 milligram preferred.

[0055] When the acrylic polymer and lecithin are combined, a matrix, ornet-like structure is formed. The ratio of lecithin to polymer areratios between 1:1000 and 1000:1. The preferred ratios of lecithin topolymer are ratios between 1:10 and 10 to 1. The relative proportions oflecithin and acrylic polymer may be found to be important to theefficiency of delivery of different antigens, i.e., bacterial, viral,parasitic or sub-units of these organisms. The optimum ratio may bedetermined by the conventional means of testing the different ratios oflecithin to polymer with the desired antigen in animals.

[0056] The adjuvant composition can be used for the delivery of vaccineantigens such as whole killed or attenuated virus, bacteria, or parasitevaccine antigens or sub-unit(s) of such organisms to mucosal surfaces,such as oral cavity, gut, nasal, vaginal and rectal surfaces. Electronmicroscopic evaluation shows that there exists a physical and/orchemical affinity between lecithin and polymer. This affinity orassociation appears as a matrix, or net-like structure. Withoutintending to be bound by any particular theory, it is believed that astructure such as this can function as a means of physically trapping orencapsulating vaccine antigen. Such binding of antigen is furtherenhanced by the electrical charge and the hydrophilic and hydrophobicproperties of lecithin and the acrylic polymers of this invention. Tofacilitate this, a polymer of different electrical charge may beselected depending on the anionic or cationic properties of the antigen.Likewise a polymer and lecithin of different hydrophobicity may beselected depending on the lipophilic or hydrophilic properties of theantigen.

[0057] If necessary or desired, the antigen can be coupled to thelecithin-acrylic polymer matrix with a cross-linker such asglutaraldehyde in a concentration of from about 1 to 50 mM, andpreferably about 15 mM. Further, the antigen can be coupled usingwater-soluble carbodiimide in a concentration ranging from about0.05-0.5 M, with about 0.1 M being preferred, or a coupling method usinga heterofunctional reagent such as N-hydroxysuccinimidyl3-(2-pyridyidithio)propionate (SPDP) in a concentration ranging fromabout 0.1-1.0 mM, and preferably about 0.2 mM. Other appropriatecoupling agents include

[0058] mixed anhydride and bisdiazotized benzidene. The cross-linker isused to improve the binding affinities of the components of the adjuvantcomposition, for example, where the components are not electricallyattracted to each other.

[0059] The strong mucoadhesive and adsorptive properties of the acrylicacid/lecithin combination also make it an excellent mechanism to aid inthe adsorption of vaccine antigen onto mucosal surfaces. The adjuvantdelivery system's absorption enhancement properties helps bring thevaccine antigen in contact with mucosal associated lymphoid tissue.Thus, an immune response is engendered that will aid in the protectionof an animal from infections and/or disease process. A robust mucosalimmune response is critical since most infectious disease-causingorganisms gain entry to the animal at mucosal surfaces. The inventioncan also be used as an adjuvant for injectable vaccines.

[0060] The vaccine comprising the adjuvant is delivered to a mucosalsurface by direct application, ingestion through the oral cavity,insertion, injection, and through other conventional means known in theart. Alternatively, the adjuvant may also be administered as aconventional injectable, which is typically either a liquid solutions orsuspension. When administered in a food or beverage carrier, theadjuvant/vaccine composition of this invention is generally included inthe carrier composition in a concentration ranging from about 0.0001-10%by weight/volume (w/v) in case of a beverage carrier and weight/weight(w/w) in case of a food carrier, with about 0.01-1.0% w/v or w/wrespectively, being preferred. When administered in an injectable, theadjuvant/vaccine composition should be present in a concentrationranging from about 0.02-2.0% by weight, with about 0.1-0.5% by weightbeing preferred.

[0061] The adjuvant/vaccine may also be administered in otherconventional solid dosage forms, such as in tablets, capsules, granules,troches, and vaginal or rectal suppositories. If administered in a soliddosage form, the adjuvant/vaccine composition should constitute between0.0001-10% by weight of the dosage form, with about 0.01-1.0% by weightbeing preferred.

[0062] In addition to the active compounds, the pharmaceuticalcompositions of this invention may contain suitable excipients andauxiliaries which facilitate processing of the active compounds intopreparations which can be used pharmaceutically. Oral dosage formsencompass tablets, capsules, and granules. Preparations which can beadministered rectally include suppositories. Other dosage forms includesuitable solutions for administration parenterally or orally, andcompositions which can be administered buccally or sublingually.

[0063] The pharmaceutical preparations of the present invention aremanufactured in a manner which is itself well known in the art. Forexample the pharmaceutical preparations may be made by means ofconventional mixing, granulating, dissolving, lyophilizing processes.The processes to be used will depend ultimately on the physicalproperties of the active ingredient used.

[0064] Suitable excipients are, in particular, fillers such as sugarsfor example, lactose or sucrose mannitol or sorbitol, cellulosepreparations and/or calcium phosphates, for example, tricalciumphosphate or calcium hydrogen phosphate, as well as binders such asstarch, paste, using, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and/orpolyvinyl pyrrolidone. If desired, disintegrating agents may be added,such as the above-mentioned starches as well as carboxymethyl starch,cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a saltthereof, such as sodium alginate. Auxiliaries are flow-regulating agentsand lubricants, for example, such as silica, talc, stearic acid or saltsthereof, such as magnesium stearate or calcium stearate and/orpolyethylene glycol. Oral dosage forms may be provided with suitablecoatings which, if desired, may be resistant to gastric juices.

[0065] For this purpose concentrated sugar solutions may be used, whichmay optionally contain gum arabic, talc, polyvinylpyrrolidone,polyethylene glycol and/or titanium dioxide, lacquer solutions andsuitable organic solvents or solvent mixtures. In order to producecoatings resistant to gastric juices, solutions of suitable cellulosepreparations such as acetylcellulose phthalate orhydroxypropylmethylcellulose phthalate, dyestuffs and pigments may beadded to the tablet coatings, for example, for identification or inorder to characterize different combination of compound doses.

[0066] Other pharmaceutical preparations which can be used orallyinclude push-fit capsules made of gelatin, as well as soft, sealedcapsules made of gelatin and a plasticizer such as glycerol or sorbitol.The push-fit capsules can contain the active compounds in the form ofgranules which may be mixed with fillers such as lactose, binders suchas starches, and/or lubricants such as talc or magnesium stearate and,optionally, stabilizers. In soft capsules, the active compounds arepreferably dissolved or suspended in suitable liquids, such as fattyoils, liquid paraffin, or liquid polyethylene glycols. In additionstabilizers may be added. Possible pharmaceutical preparations which canbe used rectally include, for example, suppositories, which consist of acombination of the active compounds with the suppository base. Suitablesuppository bases are, for example, natural or synthetic triglycerides,paraffin hydrocarbons, polyethylene glycols, or higher alkanols. Inaddition, it is also possible to use gelatin rectal capsules whichconsist of a combination of the active compounds with a base. Possiblebase material include for example liquid triglycerides, polyethyleneglycols, or paraffin hydrocarbons.

[0067] Suitable formulations for parenteral administration includeaqueous solutions of active compounds in water-soluble orwater-dispersible form. In addition, suspensions of the active compoundsas appropriate oily injection suspensions may be administered. Suitablelipophilic solvents or vehicles include fatty oils for example, sesameoil, or synthetic fatty acid esters, for example, ethyl oleate ortriglycerides. Aqueous injection suspensions may contain substanceswhich increase the viscosity of the suspension, including for example,sodium carboxymethyl cellulose, sorbitol and/or dextran. Suchcompositions may also comprise adjuvants such as preserving, wetting,emulsifying, and dispensing agents. They may also be sterilized, forexample, by filtration through a bacteria-retaining filter, or byincorporating sterilizing agents into the compositions. They can also bemanufactured in the form of sterile solid compositions which can bedissolved or suspended in sterile water, saline, or other injectablemedium prior to administration.

[0068] In addition to administration with conventional carriers, activeingredients may be administered by a variety of specialized deliverydrug techniques which are known to those of skill in the art, such asportable infusion pumps.

[0069] The lecithin/polymer adjuvant of this invention serves multiplefunctions when it is delivered orally in food and water: 1) it protectsthe vaccine antigen from degradation by the stomach acid and digestiveenzymes; 2) transports the antigen to the mucosal surfaces; 3)facilitates adsorption of the antigen onto the mucosal surfaces; 4)enhances absorption of the antigen; and 5) enhances the immune responseto the antigen due to the adjuvant properties of the two components. Inthe case of delivery to nasal, oral cavity, vaginal and rectal mucosa,the lecithin/acrylic polymer complex functions as a system to deliverand adsorb the antigen to the mucosal surface. Once adsorbed onto themucosal surface and absorbed, an immune response is engendered.

[0070] The combination of polymer and lecithin of this inventionunexpectedly provides an improved vaccine delivery system for vaccineantigens. It is apparent that the invention is also an improved deliverysystem for drugs, such as hormones, antibiotics, probiotics andantivirals. The current invention provides a more simple and efficientmethod of incorporation of antigen into a delivery system with no, orminimal damage, to vaccine epitopes. The vaccine formulation can be doneat low cost and can be easily commercialized as a feed or water additiveor as an oral paste or tablet. It is to be understood that theseformulations also would be effective in delivering antigen onto othermucosal surfaces, such as nasal, rectal and vaginal surfaces, and wouldbe effective as an adjuvant for an injectable vaccine. In addition, thehydrophobic properties that aid in the adsorption of the adjuvant andvaccine antigen to mucosal surfaces also provides a means of applying toanimal feeds, whether it be plant foliage or seeds, both which have ahydrophobic wax surface.

[0071] The composition with which the current invention is concerneddiffers from the prior art in that it comprises a mixture of lecithinand, in a preferred embodiment, an acrylic polymer or copolymer. Theinvention provides certain advantages over other vaccine deliverysystems described in the prior art. It is not prepared under harshconditions that adversely affect the substance such as the use oforganic solvents. It does not require elevated temperatures tomanufacture and does not require a stabilization step. The inventionprovides a simpler method of incorporation of antigen with minimaldamage to vaccine epitopes. Using this simpler method of manufacturingresults in low cost and ease of commercialization.

[0072] The following examples are intended to further illustrate theinvention and its preferred embodiments. They are not intended to limitthe invention in any manner.

EXAMPLE 1

[0073] Vaccine Plus Adjuvant Effectiveness

[0074] An experimental vaccine was made comprising bovine serum albuminFraction 5 (BSA) (Sigma, St. Louis, Mo.) as a non-living model antigen,lecithin (Centrolex P, Central Soya, Fort Wayne, Ind.) and an acrylicacid polymer resin (Carbopol 934P, (B F Goodrich, Cleveland, Ohio). Asecond vaccine was made comprising only BSA.

[0075] The lecithin and acrylic polymer were suspended together in 150milliliters (ml) phosphate buffered saline (PBS), each at aconcentration of 4 milligrams (mg) per milliliter (ml). The componentswere first dispersed by stirring with a magnetic stir bar and then mixedfurther in a Waring Blender using an emulsification head. The mixturewas then autoclaved to sterilize the adjuvant mixture. Bovine serumalbumin was dissolved in PBS at a concentration of 2 mg/ml and filtersterilized. One part lecithin/acrylic polymer adjuvant was then combinedwith one part of BSA. Merthiolate (0.01%) was added as a preservative.The final concentration of the vaccine components was 2 mg/ml of thelecithin/acrylic polymer and 1 mg/ml of BSA.

[0076] CF-1 female mice, approximately 18 grams, from Charles RiverLaboratories, (Willmington, Mich.), were injected subcutaneously in thegroin area with 0.1 ml of vaccine (0.1 mg. of BSA/dose) on days 0 and21. Mice were bled on day 45, 24 days after the second vaccination. Micewere bled by cutting the brachial artery following euthanasia bycervical dislocation.

[0077] Blood serum Immunoglobulin G (IgG) anti-BSA antibody titers weredetermined by an enzyme linked immunosorbant assay (ELISA). Results areshown in Table 1. TABLE 1 Results of antibody titers Reciprocal ofNumber Geometric Mean Adjuvant Group Of Mice Titer None 8 51,200Lecithin/Acrylic 8 157,916 Polymer

[0078] Results show that the adjuvant comprising a combination oflecithin and acrylic polymer does indeed enhance the immune response toan antigen.

EXAMPLE 2

[0079] Comparison of Individual Vaccine Adjuvants Administered Orally

[0080] Experimental vaccines, for delivery by the oral route, wereprepared in PBS. The vaccines comprised the antigen, BSA Fraction 5, ata concentration of 400 micrograms (μg) per ml. Vaccine 1 contained noadjuvant only BSA. Vaccine 2 was comprised of BSA mixed with 3 mg/ml oflecithin, Centrolex P. Vaccine 3 was comprised of BSA mixed with 3 mg/mlof the acrylic polymer, Carbopol 934P. Vaccine 4 was comprised of BSAmixed with 3 mg/ml of lecithin (Centrolex P) and 3 mg/ml of acrylicpolymer (Carbopol 934P). Mixing was first done with a laboratory benchtop magnetic stir bar and then in a Waring blender using anemulsification head. Lactobacillus culture was added to all vaccinesjust prior to vaccination. The final concentration of Lactobacillus was0.01 μg/ml of vaccine. On days 0, 4, 29 and 33 the groups of CF-1 femalemice from Charles-River Laboratories and weighing approximately 18grams, were administered 0.5 ml of vaccine orally by feeding needle. Onday 53, 20 days post fourth vaccination, mice were euthanized and bledby the brachial artery. End-point anti-BSA serum IgG antibody titerswere determined by ELISA. A 1/100 starting dilution of serum was useddue to non-specific background color development at dilutions less than1/100 Results are recorded in Table 2: TABLE 2 Effect of adjuvantcomposition on the anti-BSA antibody response Reciprocal of No. of MiceGeometric Adjuvant With Titer >/= Mean of Mice Composition 1/100 (%)With Titers None 3/9 (33) 158 Lecithin 4/6 (67) 141 Acrylic Polymer 6/9(67) 8,063 Lecithin and Acrylic 6/9 (67) 45,614 Polymer

[0081] Anti-BSA IgG antibody titers were over five times higher when acombination of lecithin and acrylic polymer was used as adjuvant thanwhen acrylic polymer was used alone and 323 times higher than whenlecithin was used alone. This demonstrates that the combination oflecithin and acrylic polymer is far more effective at delivering theantigen orally to the mucosal surface for uptake by lymphoid tissue thaneither lecithin or acrylic polymer alone. Although, not all of the miceshowed a serum anti-BSA IgG antibody response the results clearly show asynergistic adjuvant effect of lecithin combined with the acrylicpolymer. However, the mice that did not seroconvert may have had asecretory IgA antibody response. Indeed, oral vaccination, and mucosalvaccination in general, stimulates IgA secreting cells at mucosalsurfaces.

EXAMPLE 3

[0082] Second Test of Lecithin/Polymer Adjuvant by the Oral Route

[0083] Two vaccines were prepared in PBS that comprised the antigen, BSAFraction 5, at a concentration of 400 ug per ml. One vaccine containedno adjuvant only BSA. The other vaccine was comprised of BSA adjuvantedwith 3 mg/ml of lecithin (Centrolex P and 3 mg/ml of acrylic polymer(Carbopol 934P). The vaccine was assembled as described in Example 2. Ondays 0, 4, 27 and 31 groups of CF-1 female mice from Charles-RiverLaboratories and weighing approximately 18 grams, were administered 0.5ml of vaccine orally by feeding needle. On day 52, 21 days postvaccination, mice were euthanized and bled by the brachial artery.End-point anti-BSA serum IgG antibody titers were determined by ELISA. A{fraction (1/100)} starting dilution of serum was used due tonon-specific background color development at dilutions less than{fraction (1/100)}. Results are recorded in Table 3: TABLE 3 Comparativeresults of adjuvant versus control Reciprocal of No. of Mice withGeometric Mean of Mice Adjuvant Composition Titer >/= 1/100 (%) withTiters None 1/12 (8)  100 Lecithin and 9/11 (82) 18,812 Acrylic Polymer

[0084] This study again demonstrates that the combination of lecithinand acrylic polymer is effective in delivering antigen to oral mucosalsurfaces.

[0085] In a separate study, 4/10 mice that received this same vaccine,had a geometric mean titer of {fraction (1/1,345)} six weeks after onlya single vaccination. This demonstrates the potential of the adjuvantcomposition, when once optimized, to engender an immune response of longduration.

EXAMPLE 4

[0086] Administration of Vaccine Intranasally

[0087] Two experimental vaccines for delivery by the intranasal routewere prepared in PBS comprising the antigen, BSA, at a concentration of500 μg/ml. One vaccine was comprised of BSA alone. The second vaccinewas comprised of BSA adjuvanted with a combination of 3 mg/ml oflecithin (Centrolex P) and 3 mg/ml of the acrylic polymer, Carbopol934P. The lecithin and acrylic polymer were first mixed with alaboratory bench top magnetic stir bar and then in a Waring blenderusing an emulsification head. BSA was then added and mixed again usingthe emulsification head. Mice were vaccinated on days 0 and 20. Forty μlcontaining 20 μg of BSA antigen were placed on the nose while the mouthwas held shut. The vaccine entered the nose when the mouse inhaled. Onday 41, 21 days post second vaccination, the mice were euthanized andbled by cutting the brachial artery. Anti-BSA antibody titers weredetermined by ELISA. The starting dilution of serum was at {fraction(1/100)} due to non-specific background color development at lowerdilutions. Results are shown in Table 4: TABLE 4 Comparative results ofadjuvant vs. control Reciprocal of Adjuvant No. of Mice with GeometricMean of Composition Titer >/= 100 (%) Mice with Titers None 0/11 (0%)  0Lecithin and 7/12 (58%) 269 Acrylic Polymer

[0088] None of the mice (0/11) vaccinated with BSA alone seroconverted.The BSA antigen alone, when administered intranasally, failed tostimulate a serum antibody response in any of the mice. In contrast, 7of 12 mice, or 58%, developed serum anti-BSA IgG antibody titers as highas {fraction (1/3200)} following intranasal vaccination with BSA incombination with the invention comprised of lecithin and acrylicpolymer. The fact that not all mice seroconverted suggests that notenough, or perhaps none of the vaccine was inhaled by those mice thatdid not have an antibody titer greater than {fraction (1/100)}. Indeed,some, perhaps most, of the vaccine was observed to run off the nose orwas blown off the nose when the mouse exhaled. Still, the results ofthis study show that the invention, comprised of lecithin and acrylicpolymer, functions effectively as an adjuvant for the intranasaldelivery of a vaccine antigen.

EXAMPLE 5

[0089] Use of Adjuvant with Vaccine in Swine

[0090] The adjuvant invention comprising a combination of 2 mg/ml oflecithin (Centrolex P) and 2 mg/ml of acrylic polymer (Carbopol 934P)was used as a diluent for modified-live pseudorabies virus (ML-PRV) forswine. This adjuvant diluent and a control diluent consisting of sterilewater were used to rehydrate lyophilized (ML-PRV). The ML-PRV wasrehydrated immediately prior to vaccination. Groups of 10 weanedpiglets, 6 weeks of age, were vaccinated on days 0 and 21. Blood serumwas collected on days—2, 20, 28 and 48 for serological testing foranti-PRV serum neutralizing antibodies. The anti-PRV antibody responsesof piglets in the different vaccine groups are shown in Table 4. TABLE 5Results in pigs Geometric Mean Virus Neutralizing Antibody Titer on DaysPost Adjuvant First Vaccination Diluent Day 2 Day 20 Day 28 Day 48Non-Vaccinated 0 0 0 0 Control Water 0 4 34 21 Lecithin/ 0 6 69 52Acrylic_Polymer

[0091] This study showed that the invention comprising a lecithin andacrylic polymer combination functions as an adjuvant for a ML-virusvaccine adjuvant, in this case swine ML Pseudorabies vaccine virus. Thevirus neutralizing anti-PRV antibody titer to ML-PRV, which by itself isa very good antigen without an adjuvant and is used commercially withoutan adjuvant, was over twice as high when the lecithin/acrylic polymerwas used instead of water.

[0092] Having described the invention with reference to particularcompositions, theories of effectiveness, and the like, it will beapparent to those of skill in the art that it is not intended that theinvention be limited by such illustrative embodiments or mechanisms, andthat modifications can be made without departing from the scope orspirit of the invention, as defined by the appended claims. It isintended that all such obvious modifications and variations be includedwithin the scope of the present invention as defined in the appendedclaims. The claims are meant to cover the claimed components and stepsin any sequence which is effective to meet the objectives thereintended, unless the context specifically indicates to the contrary.

What is claimed:
 1. A method for delivering a vaccine to a human or ananimal mucosally comprising: formulating a vaccine comprising an antigenand an adjuvant comprising lecithin and an acrylic polymer; andadministering the vaccine to a mucosal surface.
 2. The method of claim 1wherein the vaccine is administered to a mucosal surface selected fromthe group consisting of oral cavity, gut, nasal, vaginal and rectalmucosal surfaces.
 3. The method of claim 1 wherein the vaccine isadministered to the human or animal by placing the vaccine in a food ora beverage.
 4. The method of claim 3 wherein the vaccine is administeredin a food or a beverage composition in a concentration of from about0.0001-5.0% by weight.
 5. The method of claim 4 wherein the vaccine isadministered in a food or a beverage in a concentration of from about0.01-1.0% by weight.
 6. The method of claim 1 wherein the vaccine isadministered in a dosage form selected from the group consisting oftablet, capsule, granule, suspension, solution, suppository, and troche.7. The method of claim 6 wherein the vaccine is administered in aconcentration of from about 0.0001-5.0% by weight.
 8. The method ofclaim 1 wherein the vaccine is administered in an injectableformulation.
 9. The method of claim 8 wherein the vaccine isadministered in a concentration of from about 0.2-2.0% by weight.
 10. Amethod for preparing an adjuvant composition comprising: hydratinglecithin and a polymer in saline or water; and mixing the lecithin andpolymer to form an adjuvant.
 11. The method of claim 10 wherein thelecithin and the polymer are mixed by placing the lecithin and thepolymer in a blender.
 12. The method of claim 10 wherein the lecithinand the polymer are mixed in the presence of surfactants.
 13. A methodaccording to claim 10 further including the step of microwaving orautoclaving the adjuvant.
 14. A method according to claim 1 wherein fromabout 0.001-10% by weight dry lecithin and from about 0.001-10% byweight polymer are hydrated.
 15. A method according to claim 14 furtherincluding the step of adding an antigen.
 16. A method according to claim15 wherein the antigen is added during the hydration step.
 17. A methodaccording to claim 15 wherein the antigen is added to the adjuvant.